MicroRNAs are derived from long chain RNA primary transcripts (pri-miRNAs) with a length of about 1000 bp which are cleaved in the cell nucleus by enzyme Drosha into miRNA precursors having a stem-loop structure with a length of about 60-80 nt. The precursor miRNAs are further cleaved into double-strand miRNAs with a length of about 18-26 nt after being transported to the cytoplasm. After the double-strand miRNAs are unwound, the mature miRNAs enter into RNA-induced silencing complexes (RISC), which are completely or not completely paired with the complementary mRNAs, so that the target mRNAs are degraded or the expression thereof is repressed.
MicroRNAs account for a small proportion of the total cellular RNA; however, miRNAs participate in a series of important processes in the life process, including early embryonic development, cell proliferation and cell death, apoptosis and fat metabolism, cell differentiation. Considering their role in gene expression regulation, and that abnormal cell proliferation, apoptosis and the like often occur in tumors, it is therefore presumed that abnormal deletion, mutation or over-expression of miRNAs will lead to the occurrence of human diseases.
Recently, increasing amounts of evidence shows that miRNAs play a very important role in inhibiting tumor cell growth, proliferation and differentiation. In the art, there is an urgent need to understand the roles of various different miRNAs to develop new medicaments for treating tumors.
RNA interference (RNAi) is a powerful experimental tool in the laboratory that uses homologous double-stranded RNAs (dsRNAs) to induce the silencing of sequence-specific target genes and rapidly block gene activity. siRNAs play a central role in an RNA silencing pathway and are guiding elements for degradation of specific messenger RNAs (mRNAs). siRNAs are intermediates in the RNAi pathway and are necessary factors for RNAi to exert its effect. The formation of siRNAs is mainly completed by regulation of Dicer and Rde-1. The dsRNAs appear in the cell due to RNA virus invasion, transcription of transposon, transcription of reverse repeats in the genome and other facotrs. The protein encoded by Rde-1 (RNAi defective gene-1) recognizes exogenous dsRNAs, and when dsRNAs reach a certain amount, Rde-1 directs dsRNAs binding to Dicer (Dicer is an endonuclease with RNaseIII activity, and has four domains: PAZ domain of the Argonaute family, type III RNase active region, dsRNAs binding region, and DEAH/DEXH RNA helicase active region) encoded by Rde-1 to form an enzyme-dsRNA complex. After cleavage of Dicer, siRNAs form, and then with the participation of ATP, key steps in RNAi interference by RNA-induced silencing complexes present in cells are assembly of RISCs and synthesis of siRNA proteins that mediate specific responses. The siRNAs are incorporated into RISCs and then fully paired with the target gene coding regions or UTR regions to degrade the target genes, and thus it can be said that siRNAs only degrade mRNAs that are complementary to their sequences. The mechanism of the above-mentioned regulation is silencing the expression of corresponding target genes through complementary pairing, and thus it is a typical negative regulatory mechanism. The recognition of target sequences by siRNAs is highly specific. Since the degradation occurs first at a central position relative to the siRNAs, these base sites at the central position are of paramount importance, and the RNAi effect would be severely inhibited once mismatches occur. As an emerging therapeutic technology, siRNA has also entered the clinical trial stage at an unprecedented speed.
K-RAS is a member of the RAS gene family and encodes K-RAS protein, and is related to the formation, proliferation, migration, spread and angiogenesis of tumors.
K-RAS protein has GTPase activity, which is activated when it binds to GTP, and inactivated when it binds to GDP. After PKC phosphorylates K-RAS mainly located on the cell membrane, such phosphorylation process renders the binding of K-RAS to the cell membrane weakened, so that its position is changed and moved to the endoplasmic reticulum, Golgi apparatus, mitochondrion and other positions. K-RAS plays a role of a molecular switch and plays an important role in many signaling pathways.
Studies have shown that about 30% of human malignancies are associated with mutations in the RAS gene, and the product generated from mutated RAS can remain activated. In leukemia, lung cancer, rectal cancer and pancreatic cancer, K-RAS mutations are common, with 30% to 35% of patients with rectal cancer having such mutations. The mutations are related to the survival, proliferation, migration, spread and angiogenesis of tumor cells. K-RAS gene is divided into mutant type and wild type. The common mutation sites are codons 12 and 13 of exon 2 and codon 61 of exon 3 in the K-RAS gene, of which there are 7 mutation hotspots: G12C, G12R, G12S, G12V, G12D, G12A, and G13V/D, and these seven kinds of mutations account for not less than 90%.
Currently EGFR-targeted drugs on the market are: gefitinib (Iressa), erlotinib (Tarceva), cetuximab (ERBITUX), and panitumumab (Vectibix). However, EGFR-targeted drugs have a poor therapeutic effect on K-RAS mutant patients, that is because K-RAS is also activated to deliver signals downstream even in the absence of EGFR signaling; therefore, the K-RAS gene status should be first detected and then medication is selected in personalized medication. If K-RAS is a mutant, use of EGFR-targeted drugs is not recommended.
Therefore, considering that if both EGFR and K-RAS pathways can be targeted at the same time, then the upstream and downstream of pathways can be simultaneously inhibited, so that EGFR-targeted drugs produce a better therapeutic effect on K-RAS mutant patients. Therefore, there is an urgent need for a targeting therapy for K-RAS gene suppression and relevant drugs, so as to address the current problems such as there had not been drugs specific for K-RAS mutation and the K-RAS mutation renders EGFR-targeted drugs ineffective.